Co-localization of 5hmC with enhancers. (a) 5hmC over enhancers in hESCs. 5hmC peak density was plotted over predicted hESC enhancers [9, 21] in 100-bp windows. (b) Verfication of 5hmC at enhancers by measuring 5hmC levels at CCGG sites. Four 5hmC peaks at enhancers and two control regions were tested. MspI cannot cut glucosylated-5hmC but is able to cut 5hmC; therefore, copy numbers of MspI + beta-glucosyltransferase (BGT) represent 5hmC levels. On the other hand, HpaII can only cut unmodified DNA; therefore, copy numbers represent 5hmC + 5mC levels. Background signal was subtracted from the copy number of each sample and then normalized to the undigested glucosylated sample. Genomic locations of tested cytosines are indicated. Error bars represent the standard deviation. (c) Histone modifications over 5hmC peaks. The overlap of 5hmC peaks with previously defined H3K4me1- and H3K27ac-enriched regions  was calculated. Random regions with the same number and size distribution as 5hmC peaks were generated and overlap with histone modifications was calculated 100 times. Error bars represent standard deviation. (d) Histone modifications over 5hmC-marked enhancers. Predicted enhancers that overlapped with 5hmC peaks were selected. The overlap of these 5hmC-marked enhancers, as well as all predicted enhancers, with previously defined H3K4me1 and H3K27ac enriched regions  was calculated. Random regions with the same number and size distribution as the enhancers were generated and overlap with histone modifications was calculated 100 times. Error bars represent standard deviation. (e) hESC-specific expressed genes significantly overlap with 5hmC peaks. hESC-specific genes were defined as genes that were expressed in hESCs (reads per kilobase of exon model per million mapped reads (RPKM) ≥ 0.5) and silent in IMR90 cells (RPKM = 0) using published RNA-seq data . *P < 0.0001.